The increased availability and identification of genes from human and other genomes has led to an increased need for efficient expression of recombinant proteins. The expression of proteins in bacteria is by far the most widely used approach for the production of cloned genes. For many reasons, expression in bacteria is preferred to expression in eukaryotic cells. For example, bacteria are much easier to grow than eukaryotic cells. More specifically, the availability of a wealth of sophisticated molecular genetic tools and thousands of mutants make E. coli, as an expression host, extremely useful for protein production. However, the high-level production of functional proteins in E. coli., especially those from eukaryotic sources has often been difficult. Inefficient translation initiation is the most common reason for poor expression (Schoner, B. E., et al., Methods Enzymol.185:94–103, 1990).
Translation initiation depends on the polynucleotide sequence of the ribosomal binding site (RBS), its distance from the initiation codon and the sequence immediately upstream of the initiation codon (Shine J. and Dalgarno L. Proc Natl Acad Sci USA. 71(4):1342–6, 1994; and Thomas D. Y., et al., Gene 19(2):211–9, 1982). However, poor initiation can occur despite the fact that an expression vector contains a good RBS from a highly expressed protein. The presence of a strong RBS along with an appropriately spaced initiation codon does not ensure efficient translation of any particular gene. One variable that continuously changes as recombinant genes are move to different expression vectors is the nucleotide sequence following the initiation codon. In fact, this part of the coding sequence can drastically affect translation. The most accepted explanation for this influence of the beginning of the coding sequence on the initiation of translation involves the absence or presence of secondary polynucleotide structures around the RBS and 5′ end of the translated sequence (de Smit M. H., and van Duin J., Proc Natl Acad Sci USA. 87(19):7668–72, 1990).
Recently, it has been reported that the downstream box (DB) polynucleotide sequence, located immediately downstream of the start codon in T7 phage gene 0.3, causes pronounced stimulation of expression when placed upstream of cloned genes, and that this effect is probably due to a stimulation of translation efficiency (Sprengart, M. L., et al., EMBO J. 15(3):665–74, 1996; and Etchegaray, J. P. and Inouye M., J Biol Chem. 274(15):10079–85, 1999). This led to the identification of a DB consensus sequence (SEQ ID NO:4), which was also found in other highly expressed genes such as ribosomal protein, elongation factor and all tRNA synthetases (Ito, K., et al., Proc Natl Acad Sci USA. 90(1):302–6, 1993), suggesting that E. coli. might use this consensus sequence to regulate gene expression. All the identified DB elements display partial complementarity to nucleotide 1467–1481 (SEQ ID NO:5) of the 16S rRNA of E. coli. Mutagenesis analysis has indicated that, increases in the level of complementarity to this region of 16S rRNA led to increased expression. Based on this observation, it has been concluded that DB sequences enhance and stabilize the interactions of the ribosome with mRNAs by base pairing to nucleotide 1467–1481 of 16S rRNA (Ito, ibid).
The two-cistron expression system has been developed to deal with translational initiation problems and has achieved some success (Schoner, B. E., et al., Proc Natl Acad Sci USA. 83(22):8506–10, 1986). This system contains a small well-translated coupler gene followed by a stop codon and then, the gene of interest, which is translated by a re-initiation process. This two-cistron approach can greatly improve the expression of target genes in a plasmid where the RBS is very poor due to inhibitory secondary structure at the 5′ end of their mRNA. However, it cannot improve the efficiency of a weak RBS sequence (Makoff, A. J., and Smallwood, A. E., Nucleic Acids Res.18(7):1711–8, 1990).
Despite these advances in the expression of recombinant proteins in bacterial hosts, there exists a need for improved methods for high-level translation initiation and higher yields for protein production.